Effects of TAML Treated
Water on Yeast Survivorship
Michael Gemmell
9th Grade
Pittsburgh Central Catholic High School
Problem
Modern technology such as factories and
water refineries have posed potential
environmental risks. Scientist have searched for
means of degrading these risks into harmless
substances.
2
TAML
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•
•
•
•
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GREEN catalyst
engineered by Dr. Terry
Collins at CMU
Accelerates H2O2
oxidation reaction
Made of common
elements found in
nature.
(C, H, O, N, Fe)
GREEN
Easy to synthesize
TAML
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Low working
concentrations
Breaks itself down after
reaction
Highly water soluble
Produces non-toxic
byproducts
Breaks down stable
chlorine compounds
Step towards a
sustainable society
Reduce exotic elements
Think in terms of
nature’s chemistry
Possible Uses
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Textiles
Water Cleaning
Petroleum Refining
Biological Warfare
TAML could be
effective in degrading
triclosan.
Triclosan
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•
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Antibacterial and anti-fungal
agent
Increasing use in consumer
products (soaps, textiles,
kitchen utensils)
Binds to ENR enzyme in cell,
which increases affinity for
NAD+ (nicotineamideadenine-dinucleotide), which
is a stable conformation of the
3 chemicals which inhibits
fatty acid synthesis.
Triclosan Health Concerns
•
•
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Can be degraded in water
by UV radiation to form
several highly-toxic
dioxins that act as potent
endocrine disruptors.
Only partially degraded
by current water
treatment methods
Significant amount is
released into environment
Increases the risk of
infertility, early puberty,
and other hormone
related problems in
humans.
Purpose
Determine the ability of TAML + H2O2 to
degrade Triclosan, and test that reaction's
residue on the survivorship of yeast.
Yeast
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Eukaryotic, unicellular fungi.
•
Used in genetics and cell biology,
in light of the fact that the cell
cycle of a yeast cell is similar to
the cell cycle of humans.
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Saccharomyces cerevisiaecommon yeast.
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Similar genetic makeup,
biochemistry, and cell cycle to
other eukaryotes, including human
cells.
•
Most studied cell in the world.
Hypothesis
Null: The residue of TAML-Activated H2O2 and Triclosan
reaction will not have a significant effect on the survivorship
of yeast.
Null: Triclosan by itself will not have a significant effect on the
survivorship of yeast cells.
Null: TAML-Activated H2O2 will not have a significant effect on
the survivorship of yeast cells.
Alternate: The residue of TAML-Activated H2O2 and Triclosan
will have a significant effect on the survivorship of yeast cells.
Materials
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Triclosan (99% pure, powdered)
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Tube Racks
Fe-TAML B catalyst
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Permanent Marker
H2O2 (.88 M)
Ethanol
Lab-Grade Catalase (Ward’s) •
Saccharomyces cerevisiae
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Matches
Sterile 250mL sidearm flask
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Vortex
NaHCO3
Sterile Water
Test tubes
YEPD Media
Bacto peptone
20 g 2%
Micropipette and sterile tips
Yeast extract 10 g 1%
Pipette pump and sterile tips
Dextrose
20 g 2%
60 Sterile YEPD Agar Petri DishesH2O to 1L
Procedure
• A culture of yeast was grown overnight in a sterile 250mL sidearm flask in LB
media at 37°C.
• The culture was left to grow until it reached an absorbance reading of about 50
Kletts, which represents a cell density of about 107 cells/mL.
• The culture was serially diluted to a concentration of approximately 103 cells/mL.
• 0.0353g of solid Fe-TAML B was added to 10mL of sterile water to create a
5*10-3M stock solution.
• 0.0289g of solid triclosan (99%) was added to 1mL of alcohol and 9mL of sterile
water to create a 1*10-2M stock solution.
• 0.89g of solid NaHCO3 was added to 10mL of sterile water to create a 1M stock
solution.
• 0.025g of solid lab-grade catalase was added to 50mL of sterile water to create a
working concentration of catalase.
Tube Contents
Control
0.01%
0.1%
Triclosa
n
1%
CRXM
0.1%
1%
10%
RXM
0.1%
1%
10%
Variable
0mL
0.001mL 0.01mL
0.1mL
0.01mL
0.1mL
1mL
0.01mL
0.1mL
1mL
Sterile
Water
9.8mL
9.8mL
9.79mL
9.7mL
9.79mL
9.7mL
8.8mL
9.79mL
9.7mL
8.8mL
NaHCO3 0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
Yeast
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
0.1mL
Total
10mL
10mL
10mL
10mL
10mL
10mL
10mL
10mL
10mL
10mL
13
Procedure
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TAML activators and H2O2 were mixed with Triclosan
Tubes were let sit for 20 min while the TAML oxidation reaction occurred.
0.1mL of catalase was added to each tube to quench the reaction, and
degrade any residual H2O2. Tubes were then left to quench for 20 min.
0.1mL of yeast (at 103 cells/mL) was then added to each tube.
Tubes were vortexed and then 0.1mL from each tube was plated 5 times,
creating a total of 60 plates.
Plates were then placed in an incubator set at 37°C overnight.
Colonies were then counted, each colony was assumed to have arisen from
one cell.
Triclosan Effects on Yeast Survivorship
P=2.33E-05
Colonies
200
150
100
50
0
0%
0.01%
0.1%
1%
Concentration
15
TAML+ H2O2 Effects on Yeast
Survivorship
Colonies
200
150
100
50
0
0%
0.1%
1%
10%
Concentration
16
TAML+H2O2 +Triclosan (Reaction
Mix) Effects on Yeast Survivorship
Colonies
200
160
120
80
40
0
0%
0.1%
1%
10%
Concentration
17
Summary of ANOVA Data Analysis
Variable
P-Value
Significance
TAML+H2O2+Triclosa P=0.477695
n
Insignificant
Triclosan
P=2.33E-05
Significant
TAML+H2O2
P=0.392405
Insignificant
Dunnett’s Test for Triclosan
Concentration t-value
T-crit value
Significance
0.01%
0.0136
2.78
Insignificant
0.1%
5.16
2.78
Significant
1%
5.17
2.78
Significant
Interpretations
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TAML+ H2O2 +Triclosan did not have a
significant effect on the survivorship of
yeast.
The TAML accelerated the oxidation of the
H2O2 and degraded the Triclosan.
The Triclosan only significantly affected the
survivorship of yeast when the concentration
exceeded 0.1%.
TAML+ H2O2 by itself did not have a
significant effect on the survivorship of
yeast.
Conclusions
• Null: The residue of TAML-Activated H2O2 and Triclosan reaction will
not have a significant effect on the survivorship of yeast.
Accepted
• Null: Triclosan by itself will not have a significant effect on the
survivorship of yeast cells.
Rejected
• Null: TAML-Activated H2O2 will not have a significant effect on the
survivorship of yeast cells.
Accepted
• Alternate: The residue of TAML-Activated H2O2 and Triclosan will
have a significant effect on the survivorship of yeast cells.
Rejected
Extensions/Limitations
Limitations
• Spread plating limited not only by human error, but by unequal
staggering.
• Limited to only one model.
• Limited to one anti-bacterial substance.
Extensions
• Testing higher concentrations of Fe-TAML and lower concentrations
of Triclosan could improve results.
• Using different bacteria could improve results.
• Test TAML on other anti-bacterial substances.
• Vary the length of the reaction and compare results.
22
References
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www.beyondpesticides.org/pesticides/factsheets/Triclosan%20cited.pd
f
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antoine.frostburg.edu/chem/senese/101/consumer/faq/triclosan.shtml
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www.sciencenetlinks.com/sci_update.cfm?DocID=178
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linkinghub.elsevier.com/retrieve/pii/S0025326X08001707
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www.ehponline.org/members/2006/114-11/innovations.html
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www.chem.cmu.edu/groups/Collins/
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www.chem.cmu.edu/groups/collins/about/about.html
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http://scienceforkids.kidipede.com/biology/cells/yeast.htm
TAML+ H2O2 +Triclosan
P=0.477695
Triclosan
P=2.33E-05
TAML+ H2O2
P=0.392405
TAML+ H2O2
Colonies
200
150
100
50
0
0%
0.1%
Concentration
1%
10%
Triclosan
Colonies
200
150
100
50
0
0%
0.01%
0.1%
Concentration
1%
TAML+H2O2 +Triclosan Reaction Mix
Colonies
250
200
150
100
50
0
0%
0.1%
Concentration
1%
10%